Isolation and characterization of antioxidant peptides from oyster (Crassostrea rivularis) protein enzymatic hydrolysates

Abstract Peptides from oysters have several bioactive functions. In this study, we identified antioxidant peptides from oysters (Crassostrea rivularis) and investigated their structure–function relationship. We used an 8 kDa molecular‐weight (MW) cut‐off membrane and semiprep reversed‐phase liquid chromatography to collect five peptides (F1–F5) and identified the highest‐abundance ion‐peak sequences AWVDY (F1), MSFRFY(F2), EPLRY(F3), RKPPWPP(F4), and YAKRCFR(F5) having MWs of 652, 850, 676, 877, and 943 Da, respectively, using ultra‐performance liquid chromatography‐quadrupole/time‐of‐flight tandem mass spectrometry. These peptides exhibited high antioxidant activities, similar to butylated hydroxytoluene, reduced glutathione, and ascorbic acid. F5 demonstrated the highest scavenging activity for DPPH radicals (IC50 = 21.75 μg/ml), hydroxyl radicals (IC50 = 18.75 μg/ml), and superoxide radicals (IC50 = 11.00 μg/ml), while F3 demonstrated the highest reducing power. Furthermore, F5 significantly protected Caco‐2 cells from H2O2‐induced oxidative damage. These results suggest that the antioxidant peptide F5 is a promising food additive that protects against oxidative damage.


| INTRODUC TI ON
Excessive free radicals and reactive oxygen species (ROS) promote damaging reactions in many cellular components, thereby creating oxidative stress, and promoting the onset of diseases such as cancer, gastric ulcers, arthritis, premature aging, inflammation, and atherosclerosis Shan et al., 2015;Suthisamphat et al., 2020). In food products, the use of synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene (BHT) is under strict regulation because of their potential health hazards and toxic effects (Mirzaei et al., 2015). Therefore, there is a great need for alternative antioxidants that are safe and exhibit high activity.
Recently, an increasing number of studies have focused on antioxidant peptides, which possess many advantages including high activity, innocuity, and easy absorption (Tadesse & Emire, 2020).
However, products of Crassostrea rivular are mostly entered into market after simple processing except for direct consumption. The economic value has not been fully exploited. The study of bioactive substances is the leading direction of marine research. Natural marine active peptides have high stability. In this study, we used C. rivularis as a source to separate antioxidant peptides from its enzymatic hydrolysates and investigated their sequence and antioxidant properties. After enzymatic hydrolysis, we used membrane-separation and semipreparative reversed-phase liquid chromatography (semiprep RPLC) to separate functional polypeptides, followed by investigation of their identity, molecular weight (MW) distribution, amino acid composition, and sequence using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatographyquadrupole/time-of-flight tandem mass spectrometry (UPLC Q-TOF MS/MS). Additionally, we evaluated the antioxidant activities of the peptides and their protective effects against H 2 O 2 -induced oxidative damage in Caco-2 cells. The relationship between the sequence and antioxidant activity of the peptides may provide a theoretical basis for the effective preparation of active polypeptides from C. rivularis.
Other reagents used in this study were of analytical or HPLC grade.

| Preparation of oyster hydrolysate by enzymatic hydrolysis
Oyster hydrolysates were prepared using an enzymatic method (Yang et al., 2019;Zheng et al., 2016). The oyster homogenates were added to deionized water at a ratio of 1:3.65 (w/v; g:ml) and incubated for 0.5 h. After adjusting the pH to 8.0, homogenate samples were hydrolyzed by alcalase (0.58%) and trypsase (0.22%) for 2 h at 55°C. The reaction mixture was then boiled for 20 min and centrifuged at 10,278 × g for 15 min. The supernatant was fractionated sequentially through three MW cut-off membranes (MWCOM), including one MF membrane (0.22 μm) and two UF membranes (200 kDa and 8 kDa, in that order). The yield of the fractions through the 8 kDa MWCOM was 31.41% ± 0.47%, and the percentage of peptides was 84.03% ± 0.85%, tested by trichloroacetic acid precipitation method (Dingess et al., 2019). The 0-8 kDa fraction was named CRRS-A, was lyophilized by freeze-drying, and stored at −20°C before use.

| Gel filtration analysis
The components of CRRS-A were isolated as described previously (Hu et al., 2021) with some modification. One gram of lyophilized hydrolysate, CRRS-A, was dissolved in 20 ml water and was isolated using a Sephadex G-25 gel filtration chromatography column (10 × 400 mm). Two milliliters of the solution was then eluted with Milli-Q water at a flow rate of 2 ml/min, and the absorbance of the eluent was recorded at 220 nm using an ultraviolet (UV) detector (APD-M20A; Shimadzu).

| MW
The MW distributions of the fractions were determined using an HPLC system (LC-20 AD; Shimadzu) with a UV detector (APD-M20A; Shimadzu) as described previously (Zhu et al., 2017). MW was evaluated on a TSK-GEL G2000SWXL column (7.8 × 300 mm, The peak-area normalization method was used to determine the MW distribution of the fractions.

| Amino acid composition
The method was slightly modified according to the previous study (Carrasco-Castilla et al., 2012). The fractions (per 200 mg) were hydrolyzed in 6 M HCl containing 0.1% phenol and incubated at 110°C in a sealed container for 22 h. After cooling, the solutions were dried by nitrogen flushing and dissolved in 1 ml 0.01 M HCl. The fractions (per 200 mg) were hydrolyzed in 5 M NaOH and incubated at 110°C in a sealed container for 22 h. After cooling, distilled water was added to the solutions to a final volume of 10 ml. Two milliliters of these solutions was then adjusted to pH 7.0 using 2.5 M HCl, followed by addition of double-distilled water to a final volume of 5 ml.
Elution was performed using solvent A (Milli-Q water) and solvent B (methanol) according to the following procedure: 0-1 min, 15% B; 1-4 min, 90% B; 4-10 min, 90% B; and 10-12 min, 15% B. The gradient elution was performed at a flow velocity of 0.2 ml/min, and data were acquired at a wavelength of 220 nm using a UV detector (G7117B; Agilent Technologies).
An accurate amino acid sequence for the purified peptides was determined using a Q-TOF MS/MS system (maXis Impact; Bruker,) equipped with an electrospray ionization source in positive mode. The molecular mass was determined by a single charged [M + H] + state in the mass spectrum. Spectra were recorded over the mass/charge (m/z) range of 50-2000. The capillary voltage was 3500 V, end-plate offset was −500 V, charging voltage was 2000 V, the nebulizer was 0.3 bar, the dry heater was set at 180°C, and the flow rate of dry gas was 4.0 L/ min. The peptides were fragmented by low-energy collision-induced separation to detect peptide fragments for de novo sequencing.

| Determination of reducing power
The reducing power was measured as described by Umayaparvathi, Meenakshi, Vimalraj, Arumugam, Sivagami, and (1) lg(MW) = − 0.0037t 2 − 0.0821t + 5.6697, R 2 = 0.9912 Balasubramanian (2014). Briefly, 1 ml of the solution was mixed with 2.0 ml phosphate buffer (0.2 M; pH 6.6) and 1 ml potassium ferricyanide solution (1%), followed by vortexing for 1 min and incubation at 50°C for 20 min. After incubation, 1 ml trichloroacetic acid (1%) was added and centrifuged at 13,360 × g for 10 min, after which, 1 ml of the supernatant was mixed with 1 ml distilled water and 0.2 ml ferric chloride (0.1%) and incubated at 50°C for 10 min. The response value of the samples was read at optical density (OD)700 nm using an ELISA reader (Sunrise-basic; Tecan, Männedorf,). Increased absorbance indicated enhanced reducing power. BHT, GSH, and V C were used as positive controls.
2.6.2 | DPPH radical scavenging activity DPPH radical-scavenging activity was estimated as described by Mirzaei et al. (2015) and Khan et al. (2018). Samples (2 ml) were added to 2.0 ml of 0.1 mM DPPH solution (sample group) or 2.0 ml ethanol (control group), and 2.0 ml ethanol was added to 2.0 ml DPPH solution for the blank group. The mixtures were then vortexed for 1 min and incubated at 37°C for 60 min in the dark, after which the absorbance was read at 517 nm (Sunrise-basic; Tecan).
Lower optical density indicated higher radical-scavenging activity.
The ability to scavenge the DPPH radical was calculated using the following equation: where K D is the rate of DPPH radical-scavenging activity (%), A B is the OD of the blank group, A S is the OD of the sample group, and A C is the OD of the control group. BHT, GSH, and V C served as positive controls.

| Hydroxyl radical scavenging activity
The effect of hydroxyl radicals was measured using the 2-deoxyribose oxidation method . The reaction mixture con- 0.20 ml of 10 mM hydrogen peroxide, 1 ml of distilled water, and 1 ml of sample solutions in a tube. The reaction was started by adding hydrogen peroxide. The reaction solution was incubated at 37°C for 1 min and the reaction was stopped by adding 0.2 ml of 0.1% hydrogen peroxide. As a blank control, the 0.3 ml distilled water was replaced with 0.3 ml phenanthroline dissolved in ethanol, 0.3 ml distilled water replaced with FeSO 4 , and 0.2 ml distilled water replaced with 0.2 ml hydrogen peroxide. As another control, 0.2 ml distilled water was replaced with 0.2 ml hydrogen peroxide. For the control tube, 1 ml distilled water was replaced with 1 ml sample solution. Sample absorbances were measured at 550 nm (Sunrise-basic Tecan,). The percentage of inhibition was computed using the following equation: where K H is the rate of hydroxyl radical-scavenging activity (U/ml), A C is the OD of the control group (distilled water replaced H 2 O 2 ), A S is the OD of the test sample (added sample), A N is the OD of the reaction without sample (distilled water used as the sample), A B is the OD of the buffer and distilled water, C S is the concentration of the H 2 O 2 solution, V is the volume of the sample used, and D is the fold-dilution of the sample. BHT, GSH, and V C served as positive controls.

| Superoxide radical scavenging activity
The rate of inhibition of superoxide radicals was determined using an anti-superoxide anion kit (Nanjing Jiancheng Bioengineering Institute,). The percentage of inhibition was calculated using the following equation: where K S is the rate of superoxide radical-scavenging activity (U/g), A C is the OD of the control group (distilled water), A S is the OD of the test sample group, A T is the OD of the standard (vitamin C liquor), C S is the concentration of the vitamin C liquor, and D is the concentration of the sample. BHT, GSH, and V C served as positive controls.

| Protective effects of F5 on Caco-2 cells damaged by H 2 O 2
Experiments were performed using Caco-2 (human colon cancer) cell lines (Sigma-Aldrich). Caco-2 cells were grown as a monolayer in minimum essential medium with Earle's balanced salt solution containing 10% FBS, 1% penicillin and streptomycin, and 1% nonessential amino acid at 37°C in a 5% CO 2 atmosphere. Complete medium was replaced every 2 days before collecting the cells with 0.25% trypsin EDTA.
Cell viability of the damaged Caco-2 cells treated with F5 and those treated with the control group was tested by 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Wang et al., 2012). The experiment involved first incubating F5 and complete medium for 4 h, and then using 10 mM H 2 O 2 to damage Caco-2 cells for 6 h on 96-well round-bottom microplates . The control group (without F5 or H 2 O 2 ) was incubated with complete medium for 10 h. The microplates were put in a 5% CO 2 cell incubator at 37°C for 4 h after adding 20 μl 5 mg/ml MTT solution. After that, the supernatant was slowly removed with pipet and the plates were washed twice with PBS; the cells were lysed by adding 200 μl DMSO to each well. The microplates were then shaken on a microplate oscillator for 15 min. The OD was recorded at a wavelength of 570 nm (Sunrise-basic; Tecan), and the percentage of cell viability was calculated using the following equation: where K V is the rate of cell viability (%); A C is the OD of the cells, MTT, and sample; A B is the OD of MTT and medium without cells; and A S is the OD of cells and MTT without sample.

| Statistical analysis
All experiments were performed in triplicate, and data are expressed as mean ± standard deviation. Differences among treatments were analyzed by one-way analysis of variance with a multiple-comparison Tukey test using SPSS software (v.18.0; SPSS Inc.,).

| Amino acid composition of F1 through F5
The amino acid compositions of F1 through F5 from the hydrolysis mixture of C. rivularis are shown in Table 1    F5 showed higher DPPH radical-scavenging activity than GSH but lower than V C at 1 mg/ml, whereas F5 showed the highest DPPH radical-scavenging activity among all other peptides and all positive controls at 10 mg/ml (93.36%). Hydroxyl radical is a type of (5)

| Sequence determination of F1 through F5
ROS produced in a Fenton reaction and capable of injuring several cellular constituents (Je et al., 2007). Figure 4c shows the hydroxyl radical-scavenging activities of F1 through F5 and the positive controls (BHT, GSH, and V C ), revealing higher activities for F1, F2, F3, and F5 than those of BHT (p < .001) and V C (p < .05) at the same concentrations. Moreover, F5 exhibited similarly high hydroxyl radicalscavenging activity as GSH, with the highest activity observed in F5 at 10 mg/ml (84.02 U/ml). Similarly, superoxide radical-scavenging activity increased in a dose-dependent manner (Figure 4d) Given these findings, we chose F5 to determine its ability to protect Caco-2 cells from H 2 O 2 -induced oxidative damage ( Figure 5).
Increasing F5 concentrations initially resulted in increased cell viabilities, followed by subsequent decreases, with the highest cell viability observed at 1.0 mg/ml F5. Compared with the control group (0 mg/ml of F5), we found that 0.10 mg/ml-100.0 mg/ml F5  (Figure 1). The five peptides, especially F5, possessed high antioxidant activities, similar to BHT, GSH, and V C (Figure 4 and Table 2).
Reducing power and DPPH, hydroxyl, and superoxide radicalscavenging activities are important indexes for measuring antioxidant capacity. Peptides with a higher reducing power have a greater ability to contribute electrons or hydrogen, therefore acting as good antioxidative agents (Je et al., 2009  . To gain insight into the relationships between antioxidant activity and peptide sequence, we evaluated the amino acid composition and sequences of the five identified peptides. Amino acid composition has an important effect on the antioxidant activity of peptides (Umayaparvathi et al., 2015). Previous reports show that the presence of hydrophobic amino acid residues is essential for antioxidant peptides (Shi et al., 2017;Umayaparvathi, Arumugam, Meenakshi, Dräger, Kirschning, & Balasubramanian, 2014;Wang et al., 2014). In the present study, F1 through F5 were collected by RPLC due to different polarities, with their respective sequences (the highest-abundance ion peak) identified as AWVDY, MSFRFY,

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest for publishing this manuscript.